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First published online 24 July 2008
doi: 10.1242/jcs.027144

Journal of Cell Science 121, 2643-2651 (2008)
Published by The Company of Biologists 2008
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Dynein and Star interact in EGFR signaling and ligand trafficking

Stanley J. P. Iyadurai1, John T. Robinson1, Lingzhi Ma1, Yungui He1, Sarah Mische1, Min-gang Li1, William Brown1, Annabel Guichard2, Ethan Bier2 and Thomas S. Hays1,*

1 University of Minnesota, Department of Genetics, Cell Biology and Development, Minneapolis, MN 55455, USA
2 Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093, USA


Figure 1
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  		Fig. 1. P2036, an allele of Star, enhances the Gl1 eye phenotype. Scanning electron micrographs of Drosophila eyes. (A) In the wild-type eye, ommatidia are arranged in an orderly fashion. (B) Eyes derived from Gl1 flies show disorganization in the arrangement of the ommatidia and bristles, giving a `rough' appearance to the eye. (C) The parental line P2036 is not distinguishable from the wild type. (D) Eyes expressing both P2036 and Gl1 are reduced in size and the general surface of the eye is very rough, showing a dominant enhancement of the Gl1 rough eye. Genotypes shown: (A) wild type +/+; +/+, (B) +/+; Gl1/+, (C) P2036/+; +/+, (D) P2036/+; Gl1/+.

 

Figure 2
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  		Fig. 2. The interaction of Star with Gl1 is dosage sensitive. The chromosomal deficiency Df(2L)S3, which removes the Star locus, enhances the Gl1 eye phenotype. (A) Gl1 flies have a mild but distinct disarrangement of ommatidia. (B) Df(2L)S3 flies are near wild type in appearance. (C) By contrast, flies expressing both the deficiency and the Gl1 mutation display an extreme rough-eye phenotype. The eye is small, narrow and very rough with a reduced number of ommatidia. (D) A recessive lethal allele of Glued, Gl1-3 shows little or no interaction with the Star allele, S1. By itself, S1 has a slightly rough eye, as shown in Fig. 3A. (E) S1 in combination with Gl1 strongly enhances the rough-eye phenotype. (F) The enhancement is reverted to mildly rough eye by the presence of a Star transgene. Genotypes shown: (A) +/+; Gl1/+, (B) Df(2L)S3/+; Gl1/+, (C) Df(2L)S3/+; +/+, (D) S1/+; Gl1-3/+, (E) S1/+; Gl1/+, (F) S1/+; P[hs-Star-HA]/Gl1.

 

Figure 3
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  		Fig. 3. Dhc alleles also interact with Star. (A) The S1 allele generates a dominant, mild, rough eye phenotype with slightly abnormal ommatidia. (B) Dhc1-1 dominantly enhances the S1 eye phenotype. The eyes are narrow, small and the eye surface is rougher. Dhc1-1/+ by itself does not have any dominant phenotypes. (C) S1 and Gl1 interact to enhance the rough eye. (D) Dhc1-1 further enhances the S1-Gl1 eye interaction. Wing phenotypes are also produced (see supplementary material Fig. S1). Genotypes shown: (A) S1/+; +/+, (B) S1/+; +/Dhc1-1, (C) S1/+; +/Gl1, (D) S1/+; Dhc1-1 +/+ Gl1.

 

Figure 4
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  		Fig. 4. Star is required for suppression of Gl1 by Dhc alleles. Epistasis tests were carried out by examining different mutant combinations using SEM. Representative examples of (A) the Gl1 eye, and (B) the Gl1 eye enhanced by S1, are shown to allow comparisons. (C) Su(Gl)77, a mutation in the Dhc locus, partially suppresses the Gl1 dominant eye phenotype. Dhc8-1 similarly suppresses the Gl1 rough eye (not shown). (D) The mutation in Star overcomes the suppression effect of the Dhc allele Su(Gl)77, and results in an enhanced Gl1 phenotype. The same result (not shown) is seen with Dhc8-1 in the presence of Gl1 and S1. (E) Su(Gl)77 in combination with Dhc8-1 completely suppresses the Gl1 eye phenotype. (F) Despite the presence of two Dhc mutations that suppress Gl1, S1 still shows enhancement of the Gl1 rough eye. Genotypes shown: (A) +/+; Gl1/+, (B) S1/+; Gl1/+, (C) Su(Gl)77 Gl1/+, (D) S1/+; Su(Gl)77 Gl1/+, (E) +/+; Su(Gl)77 Gl1/Dhc8-1, (F) S1/+; Su(Gl)77 Gl1/Dhc8-1.

 

Figure 5
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  		Fig. 5. Star cofractionates with dynein. Shown are immunoblot results using antibodies against Star-HA and dynein heavy chain (DHC). (A) A crude preparation of vesicles (V) derived from fly heads expressing HA-tagged Star (see Materials and Methods) was centrifuged at 100,000 g and the resulting supernatant (S) and pellet (P) fractions were analyzed by immunoblot. Equivalent volumes were loaded in each lane. (B) A membrane-enriched sample was fractionated on a Nycodenz step gradient. Equal volumes of each fraction were analyzed by western blot (fraction 1=bottom of gradient). The starting sample (L) and the pellet resulting from the gradient centrifugation (P) are also shown. Star and Dhc are present in overlapping fractions. (C) A vesicle membrane pellet (see part A) from hsStar-HA flies was resuspended in soluble (100,000 g) extract derived from wild-type flies. Paclitaxel (taxol) was used to promote microtubule polymerization in the absence (–) or presence (+) of ATP. In control samples, where no microtubules were assembled (–taxol, –ATP), neither dynein nor Star-HA is present in the pellet following low-speed centrifugation. In the pellets containing polymerized microtubules, Star-HA shows a greater enrichment in the absence of ATP, consistent with an interaction with dynein. The immunoblot shows pellets resulting from each experimental condition. (D) Membrane-enriched samples containing both dynein and Star-HA were prepared from S2 cultured cells by flotation on step gradients, and proteins were crosslinked with EDC for the times listed above each lane. As time progresses, increasing amounts of a very high molecular mass complex containing Dhc are detected at the top of the gel (arrowhead), with a corresponding decrease in noncrosslinked Dhc (arrow). Similarly, on a replicate blot, increasing amounts of Star-HA are seen in a very high molecular mass band (arrowhead) that coincides with the crosslinked Dhc band, while noncrosslinked Star-HA (arrow) decreases over time. Tubulin and actin, shown as negative controls, do not enter the high molecular mass complex. Note the formation of dimeric tubulin over time (~110 kDa; asterisk).

 

Figure 6
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  		Fig. 6. Dhc alleles and Gl1 enhance the dominant eye phenotype associated with Elp1. (A) Elp1 exhibits a dominant rough eye phenotype, with elliptical eyes and disarranged ommatidial arrays. (B) Dhc8-1/+ by itself does not have any dominant eye phenotype (not shown), but the combination of Dhc8-1 and Elp1 produces a reduced, narrower eye with fewer ommatidia. (D) Similarly, the combination of Gl1 and Elp1 results in a more extreme eye phenotype than that of either parent (see A and C for comparison). Genotypes shown: (A) Elp1/+; +/+, (B) Elp1/+; Dhc8-1/+, (C) +/+; Gl1 Sb/+, (D) Elp1/+; Gl1 Sb/+.

 

Figure 7
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  		Fig. 7. Gl1 suppresses the wing vein phenotypes of Elp, Dl and rho. Light micrographs of wings at low magnification (A,B) and high magnification (C-J). The L2 vein meets the upper wing margin. In comparison to wild-type (C) and Gl1 (D) backgrounds, the Elp1/+ and Dl13/+ (E and G, respectively) mutant backgrounds display an abnormal broadening of L2 at the wing margin. Gl1 in combination with either of these mutations (F,H) suppresses the wing vein phenotype. (I) Overexpression of rho in the transgenic line hsrho30A causes extra wing vein formation. (J) Gl1 suppresses the extra wing vein phenotype in hsrho30A +/+ Gl1 flies. The suppression of the wing phenotype is not completely penetrant. A majority (~70-80%) of the adults of this class exhibit the suppressed phenotype, whereas the remainder exhibit a `less mutant' phenotype. Genotypes shown: (A,C) wild type, (B) Elp1/+, (D) Gl1 Sb/+, (E) Elp1/+; +/+, (F) Elp1/+; Gl1 Sb/+, (G) Dl13/+, (H) Dl13/Gl1 Sb, (I) hsrho30A/+, (J) hsrho30A +/+ Gl1.

 

Figure 8
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  		Fig. 8. Increased levels of secreted Spitz suppress the rough eye phenotype. The rough eye caused by transgenic expression of a truncated Glued product is suppressed by sSpitz overexpression. (A) When the {Delta}Gl transgene is driven by two copies of an actin-GAL4 driver, a rough-eye phenotype results. (B) The {Delta}Gl phenotype is significantly suppressed by coexpression of the secreted form of Spitz protein from a UAS-sSpitz transgene. Genotypes shown: (A) UASp-{Delta}Gl, act5c-GAL4/CyO; act5c-GAL4/+, and (B) UAS-sSpitz/UASp-{Delta}Gl, act5c-GAL4; act5c-GAL4/+.

 

Figure 9
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  		Fig. 9. Dynein is required for vesicle formation and transport of Spitz-GFP in S2 cells. (A) Spitz-GFP distribution in S2 cells is largely restricted to the ER (see also, Tsruya et al., 2002Go). A single 1 µm section is shown to highlight the membrane network. (B) Coexpression of Spitz-GFP and Star-HA shifts the distribution of Spitz-GFP into vesicles that exhibit transient movements through the cytoplasm. A projection of sequential images shows the tracks of vesicle movements. Red bars highlight the position of several of these tracks. See also supplementary material Movie 1. (C) RNAi depletion of Dhc reduces vesicle number and inhibits motility. No tracks of moving vesicles are seen in this projection of sequential images. See also the graphs in E and F. (D) Dynein is present throughout the cytoplasmic compartment, and colocalizes with a subpopulation of Spitz-GFP vesicles after coexpression of both Spitz-GFP and Star-HA. An image stack of four optical sections shows both Dhc (red) and Spitz-GFP (green) channels. Arrows highlight the positions of some of the overlapping signals, which appear yellow. The inset is enlarged by a factor of two. Scale bar: 10 µm (applies to all images). (E) RNAi depletion of Dhc decreases the number of Spitz-GFP vesicles per cell. Graph shows the average number of Spitz-GFP vesicles observed in a single focal plane. Error bars depict ± s.e.m. Wild type, 339 vesicles from 9 cells; Dhc RNAi, 356 vesicles from 25 cells (P<0.001). (F) Depletion of Dhc also decreases the frequency of transport events of Spitz-GFP vesicles. The graph shows the average percentage of vesicles in each cell that are motile, calculated from the same cells used in E. Error bars depict ± s.e.m.

 

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© The Company of Biologists Ltd 2008